Spectrin-actin junctions play critical roles in both the maintenance of red blood cell shape and membrane properties. Their organization and linkage to the cell membrane is therefore of fundamental importance to erythrocyte physiology. Our recent evidence indicates that dematin performs its function by linking spectrin-actin junctions to the cell membrane. Here, we propose that dematin links spectrin-actin junctions to the cell membrane via the glucose transporter, GLUT1, in human erythrocytes and via a related transporter in mouse erythrocytes. We will test this hypothesis with the following three aims. A1: Characterization of GLUT1 interactions with spectrin-actin junctions in human erythrocytes. Deletion of the headpiece domain of dematin results in a compensated anemia with microcytosis and spherocytosis in mice. We identified GLUT1 as the primary membrane receptor for both dematin and adducin in human erythrocytes. We will identify their interacting domains and the critical sequence elements and, through disruption of the GLUT1-cytoskeletal bridge by biochemical means, assess its functional impact on membrane stability and thus the shape of human erythrocytes. A2. Identification of dematin- and adducin-binding receptor(s) in mouse erythrocytes and adipocytes. The GLUT1 receptor does not link spectrin-actin junctions to the membrane in mouse erythrocytes. We predict that dematin and adducin bind to a novel membrane receptor(s) in mouse erythrocytes. We will identify this membrane receptor using a series of biochemical and proteomic approaches. We will determine whether dematin and adducin bind to GLUT4, the insulin-sensitive glucose transporter generally found in adipose and muscle cells, and investigate their role in localization and recycling of GLUT4 in adipocytes. These experiments will clarify the role of alternate receptors that link the spectrin-actin junctions in mouse erythrocytes and adipocytes. A3. Determination of the effects of complete dematin deficiency. As the core domain of dematin is still expressed in headpiece-null mice, there remains the possibility that more profound hematological and metabolic phenotypes develop in mice carrying a homozygous gene disruption of full-length dematin. We propose to generate the latter mouse model and conduct a comprehensive analysis of the hematological and metabolic lesions, focusing particularly on GLUT4 trafficking and the potential development of diabetes. Together, these studies will elucidate the specific role(s) of the headpiece and core domains of dematin in erythropoiesis and intermediary metabolism. PUBLIC HEALTH RELEVANCE: Erythrocyte membrane has served as a paradigm for discovering the function of its proteins in many non-erythroid cells. This project will investigate the role of dematin, adducin, and glucose transporters in the formation of a novel membrane-cytoskeletal bridge with functional implications in hemolytic anemia, diabetes, and obesity.